The present invention relates to a cytotoxicity testing method and, more particularly, to a method of quantizing the toxicity of a chemical substance by counting live cells and dead cells.
The object of toxicity tests for chemical substances is to estimate the danger of the substances to human beings. For such tests, use has customarily been made of higher animals including Mammalia and lower Vertebrata including fishes. Today, however, toxicity tests using animals are being replaced with cytotoxicity tests using the cultured cells of higher animals.
Conventional cytotoxicity testing methods will hereinafter be described with reference to "Cytotoxicity Testing Methods", the Institute of Tissue Culture Engineers of Japan, 1991, Asakura Publishing Company. A first testing method uses the obstruction of the growth of cells as an index. Cells reached a logarithmic growth stage are implanted in a plastic laboratory dish and then cultured for 24 hours in a 37.degree. C. carbon dioxide incubator. After the cells have fully adhered to the dish and again started to grow, a chemical substance to be tested and diluted by a culture fluid such that the final content will coincide with a preselected content is directly added to the culture fluid. On the elapse of 24 hours, the culture liquid containing the chemical substance is discharged, and then a fresh culture liquid is added. In this condition, the cells are further cultured in the 37.degree. C. carbon dioxide incubator. The dish is picked out just after the addition of the chemical substance, at the end of the above treatment of the chemical substance, and on the first day, second day, third day, fifth day, seventh day and ninth day after the treatment in order to count the cells by use of a hemocytometer or an automatic cell measuring device by way of example. A cell growth curve is produced in a graph in which the abscissa and ordinate respectively indicate the number of days of culturing and the number of cells for a single laboratory disk. Numerical values representative of a period of time necessary for the number of cells to be doubled (cell doubling time) and a cell saturation density are read on the graph. The influence of the chemical substance is quantized on the basis of such numerical values in order to estimate the cytotoxicity of the substance.
A second cytotoxicity testing method uses a monolayer culture cell densitometer. After cells grown on a laboratory dish have been dyed by Crystal Violet, the degree of absorption of light having a wavelength of 555 nm by the dish is measured by the densitometer so as to determine the degree of growth of the cells. Specifically, cells are dispersed on a dish and cultured for 3 days in a carbon dioxide incubator. After a chemical substance to be tested has been added to a culture fluid in the dish, it is treated for 2 days, and then the culture fluid is discharged. After the cells have been fixed by an aqueous solution of formaldehyde, the cells are dyed by a Crystal Violet solution. After the dish has been dried by air, the degree of growth of the cells is measured by the above densitometer. For a control light absorption degree of 100%, the content of the chemical substance showing a light absorption degree of 50% is determined. The second method is often used to set the content of a substance to be treated in a chromosomal aberration induction test meant for a chemical substance.
A third cytotoxicity testing method uses Neutral Red to be taken into cells. Cells are implanted in the holes of a 96 hole microtiter plate together with a culture fluid and cultured for 48 hours in a 37.degree. C. carbon dioxide incubator. Then, a culture fluid in which a chemical substance to be tested is dissolved is added, and 48 hours of culturing is effected. Subsequently, a Neutral Red solution is added, and 3 hours of culturing is effected. After excess Neutral Red has been discharged, the cells are fixed by an aqueous solution of formaldehyde. Neutral Red taken into the cells are extracted by a 50% ethanol and 1% acetic acid solution. The degree of absorption of light having a wavelength of 540 nm by the extracted liquid is measured by a microreader. Because Neutral Red passes only through the walls of live cells and deposits on lysosome, only live cells can be specifically dyed. A graph is produced in which the abscissa and ordinate respectively indicate the content of the chemical substance tested and the ratio of the amount of dye taken into the cells treated by the substance to the amount of dye taken into the cells not treated. The degree of toxicity is quantized as a chemical substance content setting up a ratio of 50% of the amount of dye taken into the treated cells to the amount of dye taken into the non-treated cells (LD.sub.50 values).
A fourth cytotoxicity testing method uses Crystal Violet and is, in principle, substantially the same as the second method using the monolayer culture cell densitometer. Specifically, cells are sown in the holes of a 96 hole microtiter plate together with a culture liquid, and then a culture liquid containing a chemical substance to be tested is added. The cells are treated by the chemical substance for 3 days in a 37.degree. C. carbon dioxide incubator. Subsequently, a glutaraldehyde solution is added to the holes of the above plate in order to fix the cells. After the fixing solution has been discharged, a Crystal Violet solution is introduced into the holes in order to dye the cells. After the dye liquid has been discharged, the plate is washed and then dried by air. A 96 hole microplate reader is used to measure the absorption of light having a wavelength of 590 nm by the dyed cells. For a control light absorption degree of 100%, the content of the chemical substance showing an absorption degree of 50% (IC.sub.50) is determined.
A fifth cytotoxicity tensing method is a DNA assay method using a fluorescent dye. Grown cells are dyed by 2-diamidino-phenylindole (DAPI) or Hoechst 33342 which is a fluorescent dye. The fluorescence intensity of the resulting dyed cells is measured by a fluorescence photometer. Cells are sown in the holes of a 96 hole microtiter plate together with a culture fluid and cultured for 2 days in a 37.degree. C. carbon dioxide incubator. After the culture fluid has been replaced with a new culture fluid containing a chemical substance to be tested, the chemical substance is caused to act on the cells for 3 days. Subsequently, the culture fluid is discharged, and then the cells are fixed by a glutaraldehyde solution. Thereafter, the cells are dyed by DAPI or Hoechst 33342 solution. The fluorescence intensity of the resulting dyed cells is measured by the above photometer. Finally, a cell density is determined on the assumption that a control fluorescence intensity is 100%.
A sixth cytotoxicity testing method forms colonies. Usually, cultured cells form irregular colonies in which cells high in viability and growability and cells low in the same exist together. Even cells with low viability can exist in a colony when cultured together with cells with high viability, because a culture environment is prepared by the surrounding cells. The sixth method uses the colony forming ability of cells as an index and is extensively applied to the measurement of the lethal effect of radiation or that of chemical substances. Specifically, cells reached a logarithmic growth stage are implanted in a laboratory dish together with a culture fluid and cultured in a 37.degree. C. carbon dioxide incubator. After the cells have fully adhered to the dish and again started to grow, a culture liquid in which a chemical substance to be tested and having a preselected content is dissolved is added. On the elapse of a preselected period of time, the entire fluid is discharged and replaced with a new culture fluid. After the cells have been further cultured for 12 days to 14 days, the culture fluid is discharged. After cleaning and drying, the cells are fixed by methanol. The fixed colonies are dyed by a Giemsa dyeing liquid and counted. A survival rate curve is produced in a graph in which the abscissa and ordinate respectively indicate the content of the chemical substance to be tested and the logarithm of the ratio (survival rate) of the number of colonies on the treated dish to the number of colonies on a non-treated dish. Toxicity is quantized on the basis of the content of the chemical substance which lowers the survival rate to 50% or 37% (LD.sub.50 value and D.sub.0 value).
A seventh cytotoxicity testing method selectively dyes only dead cells (whose walls have some injury) and counts the number of dyed cells and that of non-dyed cells (live cells or cells whose walls are free from fatal injury), thereby determining the ratio of survived cells. Specifically, after cultured cells have been exposed to a chemical substance to be tested for a preselected period of time, they are dyed by a Trypan Blue solution. The cells whose cytoplasm and nuclei are entirely dyed in blue and the other cells are distinguished and counted by a hemocytometer individually. A survival rate is determined by: ##EQU1##
Trypan Blue may be replaced with Eosine, erythrosine, Methylene Blue, Congo Red, Nigrosine or similar dye also capable of selectively dyeing dead cells.
An eighth cytotoxicity testing method is a .sup.51 Cr isolation method. Specifically, after cells have been implanted in a culture flask, a 10 .mu.Cia aqueous solution of Na.sub.2.sup.51 CrO.sub.4 is added. The cells are held for 20 to 24 hours in a 37.degree. C., 95% air and 5% carbon dioxide incubator. As a result, .sup.51 Cr is taken into the cells, and the .sup.51 Cr index of the cells is completed. A .sup.51 Cr index cell solution is introduced into the holes of a 96 hole microplate storing a chemical substance to be tested beforehand. Then, the cells are held for 4 to 24 hours in a 37.degree. C., 95% air and 5% carbon dioxide incubator. After an absorbing material has been placed on the microplate, it is inserted into the holes by an exclusive press in order to absorb only the top. After the absorbing material has been transferred to a vial, radioactivity is measured by a gamma counter. A .sup.51 Cr isolation ratio of the cells is determined by: ##EQU2##
The above g.sup.51 Cr isolation ratio is used as the index of toxicity of the chemical substance. In the Eq. (2), the negative control is one to which a culture fluid not containing the chemical substance to be tested is added in order to measure the natural isolation of .sup.51 Cr from injury-free live cells. The positive control is one to in which a culture solution containing 0.8% of phenol or 0.5% to 1.0% of Triton X-100 or similar surfactant is added to the cell culture fluid.
A ninth cytotoxicity testing method is a .sup.51 Cr take-in method. While the above 51Cr isolation method is effective with chemical substances of the kind inducing cytolytic-death in a short period of time, it is low in sensitivity to substances of the kind not fully destroying cell walls. The .sup.51 Cr take-in method is an improved version of the .sup.51 Cr isolation method as to decision on live/dead cells. Specifically, after a cell solution has been introduced in the holes of a 96 hole microplate, a chemical substance solution to be tested is added. The plate is held at 37.degree. C. for 1 hour to 8 hours, and then the top of the solution is removed by a centrifugal force. A culture fluid containing 1 .mu.Ci of Na.sub.2.sup.51 CrO.sub.4 is introduced into the holes of the plate and held at 37.degree. C. for 30 minutes. After .sup.51 Cr not taken into the cells has been fully discharged, 50 .mu.l of acetic acid is added to each hole and fully stirred. All the contents of the holes are transferred to gamma counter tubes in order to measure their radioactivity. Cytotoxicity is calculated as follows: ##EQU3##
A tenth cytotoxicity testing method determines whether cultured cells are alive or dead by flow cytometry. When a Trypan Blue solution is added to a cell solution, only deal cells take it thereinto. Trypan Blue absorbs red helium-neon laser light having a wavelength of 632.8 nm and issuing from a flow cytometer. Therefore, a sample consisting of a number of cells is instantaneously divided into three groups of spots, i.e., live cells, dead cells, and cell fractions. The three groups of spots are drawn on an oscilloscope and allow a ratio between live cells and dead cells to be easily determined. When Hoechst 33342 and propidium iodide are used to dye DNA of unfixed cells, live cells and injured cells fluoresce in blue and red, respectively and can therefore be easily distinguished by a flow cytometer.
An eleventh cytotoxicity testing method is an agar diffusion method using agar culturing. With this method, it is possible to test the toxicity of a chemical substance without regard to its form, i.e., solid, powder, film, paste, liquid or the like. Cells are introduced into a laboratory dish together with a culture fluid and cultured for 24 hours in a 37.degree. C. carbon dioxide incubator. After cleaning using a buffer liquid, the cell layer is covered with a culture solution to which Iw/V % of agar has been added, and then left for 20 minutes to 30 minutes to turn out a gel. After dyeing using Neutral Red, the excess dyeing liquid is removed. A chemical substance to be tested is put on the agar gel, and then the dish is turned upside down and cultured for 24 hours in the carbon dioxide incubator. The distances between the end of the chemical substance and decolored cells are measured by slide calipers or a phase contrast microscope in order to determine zone indices. The distances between the end of the chemical substance and non-destroyed cells are measured by the phase contrast microscope and used as lysis indices. The degrees of injury of the cells are estimated on the basis of the two index values (0 through 5).
Some improved versions of the agar diffusion method are available, as follows. An FDA (fluoresceindiacetate) dyeing method uses FD dyeing in places of Neutral Red dyeing. A collagen gel culture method uses collagen derived from animal tissue in place of agar. A Crystal Violet/SDS extraction method uses Crystal Violet dyeing in place of Neutral Red dyeing in order to improve the quantization of the agar diffusion method. Specifically, after cells have been treated by a chemical substance to be tested, Crystal Violet is extracted from the cells by 1% SDS (sodium dodecyl sulfate). Then, cytotoxicity is quantized on the basis of the degree of absorption of light whose wavelength is 598 nm.
A twelfth cytotoxicity testing method is an LDH method. LDH which is a lactic acid dehydrogenase or lysosome is released from cells to a culture fluid due to the wall injury of the cells. The LDH method examines cytotoxicity by using LDH activity as an index and is common to the dye exclusion method and .sup.51 Cl isolation method as to wall injury. Specifically, cells are introduced in the holes of a 24 hole dish together with a culture fluid and cultured for 48 hours in a 37.degree. C. carbon dioxide incubator. After cleaning, a culture fluid containing a chemical substance to be tested is introduced into the holes and cultured for 4 hours. Thereafter, the cells and culture liquid are collected from the holes and then put in a centrifugal separator in order to measure the LDH activity of the top. The total LDH activity is corrected by naturally released LDH activity. The toxicity of the chemical substance is determined by producing an LDH release ratio, as follows: ##EQU4##
A thirteenth cytotoxicity testing method uses a millipore filter and is a simple method for testing the cytotoxicity of a chemical substance implemented as a solid or a paste. A millipore filter is put in a laboratory dish, and then cells and a culture fluid are evenly dispersed on the filter and cultured for 24 hours in a 37.degree. C. carbon dioxide incubator. After the culture fluid has been discharged from the dish, the filter is cleaned. Subsequently, the filter is turned upside down and for 24 hours in a 37.degree. C. carbon dioxide incubator. After the culture fluid has been discharged from the dish, the filter is cleaned. Subsequently, the filter is turned upside down and put on flat agar prepared on a dish with its cell surface facing downward. A chemical substance to be tested is put on the filter and cultured for 2 hours in the carbon dioxide incubator. After the removal of the chemical substance, the filter is picked up from the agar. Succinic acid dehydrogenase reaction substrate solution is introduced into a dish, and then the filter is immersed in the solution with its cell layer facing upward and held at 37.degree. C. for 3 hours. Thereafter, the filter is picked up from the dish, immersed in a formaldehyde solution, washed, and then dried. The cytotoxicity of the chemical substance is determined on the basis of a distance between the end of the substance and a non-dyed cell region.
A fourteenth cytotoxicity testing method uses MTT ([3-(4, 5-dimethylthiazol-2-yl )-2, 5-diphenyl tetrozorlium bromide]). MTT turns out formazan due to an enzyme present in intracellular mitochondria. After formazan has been dissolved in hydrochloric acid-isopropanol, calorimetric quantization is effected. The amount of formazan is dependent on the number of live cells and therefore representative of the same. Cells, a culture fluid and a chemical substance solution to be tested are introduced into the holes of a 96 hole microplate and cultured for 24 hours in acid-isopropanol solution, black crystals of formazan are dissolved by stirring. The degrees of absorption of 570 nm light and 630 nm light are determined by a microplate reader in order to quantize the toxicity of the chemical substance.
A fifteenth cytotoxicity testing method uses the obstruction effect against the migration of a radioactive precursor into cells, i.e., uses the inhibition of the synthesis of nucleic acids, proteins and so forth and sugar metabolism as an index. Migration of .sup.3 H-thymidine into DNA fractions or migration of .sup.3 H-uridine into RNA fractions is often used. As for the inhibition of protein synthesis, use is made of migration of .sup.3 H- or .sup.14 C-leucine or .sup.35 S-methionine. As for the inhibition of intercellular substrate formation, use is made of migration of .sup.35 SO.sub.4 into glucose aminoglycan fractions or migration of .sup.3 H-proline into collagen fractions. Further, as for the inhibition of sugar metabolism, use is made of migration of .sup.14 C-glucose.
How .sup.3 H-thymidine is taken into cells will be described by way of example. A cell solution is introduced into the holes of a 96 hole microplate and cultured for 2 hours in a 37.degree. C. carbon dioxide incubator. A chemical substance to be tested is added to each hole, and 4 hours of culturing is executed. After a culture solution containing 100 KBq/ml to 120 KBq/ml of .sup.3 H-thymidine has been added to each hole, 2 hours of culturing is effected in the incubator. After each hole has been evacuated and cleaned, the cells are peeled off from the plate by trypsin treatment and collected on a glass fiber filter. The filter is treated by ice-cooled 5% TCA in order to cause DNA to precipitate on the filter. The filter is cleaned, dried, and then put in a vial. After the addition of a scintillation cocktail, radioactivity is measured by a liquid scintillation meter. A ratio of the radioactivity of the cells treated by the chemical substance to a control is determined in order to quantize the toxicity of the chemical substance.
A sixteenth cytotoxicity testing method limits the amount of proteins and is a traditional method for the cytotoxicity test of anticancer medicine. A cell solution is filled in two test tubes, and then a chemical substance to be tested is introduced into the tubes. One of the tubes is put in a 37.degree. C. carbon dioxide incubator for 72 hours for culturing. The other tube is put in a centrifugal separator in order to remove the culture fluid and then cleaned, and the resulting precipitated cells are held at 4.degree. C. The tube subjected to 72 hours of culturing has also its cell layer cleaned. The amounts of proteins in the two tubes are quantized by colorimetric quantization on the basis of the degree of absorption (OD) of 650 nm light. A cell growth limitation ratio (%ICG) is produced by: ##EQU5## Ratios below 15%ICG are representative of "nonpoisonous", ratios of 15%ICG to 20%ICG are representative of "weakly poisonous", and ratios above 29%ICG are representative of "poisonous".
Japanese Patent Laid-Open Publication No. 7-123999 teaches another testing method using a base having a plurality of surface portions different in the easiness of adhesion of adhering cells. Cells, a culture fluid and a chemical substance to be tested are put in a container containing the above base and cultured. On the elapse of a preselected period of time, the base is observed in order to determine the toxicity of the chemical substance on the basis of the arrangement of the cells adhered to the base.
However, with the conventional cytotoxicity testing methods, particularly one determining the survival rate of cultured cells, it is impossible to directly or accurately count the prohibitive number of cohered live cells or dead cells one by one.
In practice, after live cells or dead cells have been colored by a dye, the degree of absorption of light whose wavelength is particular to the dye is measured in order to estimate the density of live cells or dead cells. However, this cannot be done unless a relation between the degree of light absorption by dyed cells and the actual cell density is determined beforehand. Moreover, the degree of light absorption by dyed cells does not provide a true cell density.
Technologies relating to the present invention are also disclosed in, e.g., Japanese Patent Laid-Open Publication NO. 63-295963.